含编码人精子膜蛋白cDNA的重组沙门氏菌的构建

ＨＳＤ Ｉ是编码人精子膜蛋白（ｈＳＭＰ １）的ｃＤＮＡ，将其中５５０ ｂｐ 的目的ｃＤＮＡ与编码通用终止密码子的Ａｄａｐｔｅｒ相连接，再将该重组ｃＤＮＡ插入到ｐＵＣ１９的多克隆位点，经酶切、测序鉴定后，利用ＥＣｏＲＩ酶切重组质粒，回收插入片段，并将其准确地连接到两种不同拷贝数的ａｓｄ＋ 载体（ｐＹＡ２９２、ｐＹＡ３１３７）中，使其在减毒的Δｃｙａ、Δｃｒｐ、Δａｓｄ 基因缺失的沙门氏菌（χ４５５０）中表达，Ｗｅｓｔｅｒｎ ｂｌｏｔ检测表明，两种重组沙门氏菌的全细胞裂解液与ｈＳＭＰ １的兔抗血清在２１ ＫＤ处有一明显的结合带。我们成功地构建了表达ＨＳＤＩ（５５０ ｂｐ）的两种重组沙门氏菌，并检测了其体外生长特性。该重组沙门氏菌有望成为一种新的粘膜免疫避孕的疫苗。

The Expression of the Human Sperm Membrane Protein in the Recombinant of Salmonella Typhimurium

A 550 bp cDNA fragment of HSDI coding for part of human sperm membrane protein (hSMP1) was ligated with an Adapter containing the universal stop codon,and the ligated fragment cDNA was then cloned into the MAS of pUC19.The desired plasmid with exact open reading frame was obtained,and was cut with EcoR I.The insert was purified and then cloned into the two asd + Salmonella expression vectors (the low copy number plasmid-pYA292 and the high copy number plasmidpYA3137).The recombinant plasmid with the correct orientation was selected by restriction enzyme digestion.The recombinant plasmids were transfered into the nonpathogenic Δasd Salmonella typhimurium χ4550,which has deletions of Δcya,Δcrp,Δasd genes,Westernblot analysis of the whole cell lysate of the two recombinant S.typhimurium showed a predominate protein band at 21 KD which reacted with the antihSMP1 antiserum.Two recombinant S.typhimurium containing the 550 bp cDNA of HSDI have been constructed and the characteristic of their growth in vitro were determined.They may be a new mucosal immunization antifertility vaccine.